Guanosine derivatives and use thereof

ABSTRACT

It is intended to provide a novel monomer unit by which Z type DNA can be more effectively stabilized, a reagent for integrating this monomer unit into an oligonucleotide, and a method of stabilizing Z type DNA by using the reagent. Namely, a guanosine derivative represented by the following general formula [1]: 
                         
wherein R 1  represents acyl; R 2  represents lower alkyl; R 3  represents tri-substituted silyloxy or tetrahedropyranyloxy; and R 4  represents cyanoethyl or allyl; a reagent for stabilizing Z type DNA which contains the guanosine derivative; and a method of stabilizing Z type DNA by using the guanosine derivative. It is also intended to provide a method of transferring guanosine having lower alkyl at the 8-position into an oligonucleotide by using the guanosine derivative; and an oligonucleotide carrying guanosine having lower alkyl at the 8-position transferred thereinto.

TECHNICAL FIELD

The present invention relates to novel guanosine derivatives useful as areagent for stabilizing Z-form DNA, and a method for stabilizing Z-formDNA by using the protein.

BACKGROUND ART

Although Z-form DNA is a nucleotide found about 30 years ago, which hasa left-handed Z form structure, biological roles of the Z-form DNA havenot been well established. The reason is because it is difficult toobtain a stable left handed Z-form of nucleotide in a solution in thecase of nucleotides used for studies, which usually have such a shortlength as 6 to 20 mers. Consequently, development of a monomer unit forstabilizing the Z-form DNA is demanded for studying the Z-form DNA.

The present inventors have previously found and reported in literaturesthat Z-form DNA can be stabilized by 8-methylguanosine (MeG) (e.g. referto the prior art reference 1). They have also made numerous studies byusing 8-methyldeoxyguanosine (e.g. refer to prior art references 1, 2, 3and 4).

By using 8-methyldeoxyguanosine, stabilization of the Z-form DNA, whichhas been extremely difficult to obtain up to now, could be achieved tosome extent. However, it is still insufficient to completely stabilizethe Z-form DNA, and a further study to search a monomer unit toeffectively stabilize the Z-form DNA is still demanded.

Prior art references relating to the present invention are as follows,and they are incorporated herein by reference.

-   1. Sugiyama, H.; Kawai, K.; Matsunaga, A.; Fujimoto, K.; Saito, I.;    Robinson, H.; and Wang, A. H.-J., “Synthesis, Structure and    Thermodynamic Properties of 8-Methylguanine-Containing    Oligonucleotides Z-DNA under Physiological Salt Conditions.”;    Nucleic Acid Res., 1996, 24, 1272.-   2. Kawai, K.; Saito, I.; Sugiyama, H., “Conformation Dependent    Photochemistry of 5-Halouracil-Containing DNA: Stereospecific    2′-α-Hydroxylation of Deoxyribose in Z-form DNA.”; J. Am. Chem. Soc.    1999, 121, 1391-1392.-   3. Kawai, K.; Saito, I.; Kawashima, E.; Ishido, Y.; Sugiyama, H.,    “Intrastr and 2′-β-Hydrogen Abstraction of 5′-Adjacent Deoxy    guanosine by Deoxyuridin-5-yl in Z-form DNA”; Tetrahedron Letters    1999, 40, 2589-2592.-   4. Oyoshi, T.; Kawai, K.; Sugiyama, H., “Efficient C2′    α-Hydroxylation of deoxyribose in Protein-Induced Z-form DNA.”; J.    Am. Chem. Soc., 2003, 125, 1526-1531.

DISCLOSURE OF INVENTION

The present invention has been completed under the above circumstance,and an aspect of the present invention is to provide a novel monomerunit, which can stabilize Z-form DNA more effectively, a reagent forintegrating the monomer unit to oligonucleotide, and a method forstabilizing the Z-form DNA by using the reagent.

The present invention relates to a guanosine derivative represented bythe following general formula (I):

wherein, R¹ represents an acyl group, R² represents a lower alkyl group,R³ represents a tri-substituted silyloxy group or a tetrahydropyranyloxygroup, and R⁴ represents a cyanoethyl group or an allyl group.

Further, the present invention relates to a reagent for stabilizingZ-form DNA comprising said guanosine derivative.

Further, the present invention relates to a method for stabilizingZ-form DNA by using said guanosine derivative.

Further, the present invention relates to a method for integratingguanosine having a lower alkyl group at the position-8 into anoligonucleotide, wherein said guanosine derivative is used.

Further, the present invention relates to an oligonucleotide integratedwith guanosine having a lower alkyl group at the position-8.

Namely, as a result of intensive study to solve the above problems, thepresent inventors have found that 8-methylguanosine, which is guanosineintroduced with a methyl group at the position-8, stabilizes Z-form DNAmore intensively by several times compared with the abovementioned8-methyldeoxyguanosine (MeG), and completed the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows circular dichroism (CD) spectra of three types ofoligonucleotide of the present invention obtained in Example 3.

BEST MODE FOR CARRYING OUT THE INVENTION

In the general formula [1], the acyl group represented by R¹ may be anyacyl group so long as it is usually used in this field as a modifyinggroup for amino acid, and preferably includes a bulky acyl group such asan isobutyl group, a benzoyl group and a 4-(t-butyl)benzoyl group.

The lower alkyl group represented by R² preferably includes a loweralkyl group having 1 to 4 carbon atoms such as methyl, ethyl, n-propyl,isopropyl, n-butyl, s-butyl and t-butyl group, and particularlypreferably a methyl group from the viewpoint of easiness in synthesis.

In the above general formula [1], the tri-substituted silyloxy grouprepresented by R³ preferably includes a bulky tri-substituted silyloxygroup such as a t-butyldimethylsilyloxy group, a triisopropylsilyloxygroup and a triphenylsilyloxy group.

The guanosine derivatives of the present invention represented by theabove general formula [1] are quite useful as a monomer unit forstabilizing Z-form DNA.

Namely, the Z-form DNA can be intensively stabilized using the guanosinederivatives of the present invention represented by the above generalformula [1].

In more detail, by using the guanosine derivatives of the presentinvention, Z-form DNA can be stabilized by integrating guanosine havinga lower alkyl group at the position-8 in DNA by means of DNA solid-phasesynthesis.

In this connection, various oligonucleotides, to which 8-methylguanosinewas integrated by using the guanosine derivative of the presentinvention represented by the general formula [1], wherein R¹ was anisobutyryl group, R² was a methyl group, R³ was at-butyldimethylsilyloxy group and R⁴ was a cyanoethyl group, extremelylowered midpoint of the B-Z transition as compared witholigonucleotides, to which 8-methyldeoxyguanosine was integrated by thesimilar procedures. From these results, it has been found thatoligonucleotides integrated with the guanosine derivatives of thepresent invention have a stronger stabilizing effect than those with thedeoxyguanosine derivative of the prior art by several to several dozentimes.

A process for producing the guanosine derivative of the presentinvention is illustrated in the following reaction scheme byexemplifying the guanosine derivative of the general formula [1],wherein R¹ is an isobutyryl group, R² is a methyl group, R³ is at-butyldimethylsilyloxy group and R⁴ is a cyanoethyl group.

In the above reaction scheme:

-   process a): reaction with trimethylsilyl chloride, subsequently with    isobutyric anhydride in pyridine;-   process b): reaction with H₂SO₄, FeSO₄ and t-butyl hydroperoxide in    aqueous medium;-   process c): reaction with dimethoxytrityl chloride, triethylamine    and 4-dimethyaminopyridine in pyridine;-   process d): reaction with imidazole and 2′-t-butylmethylsilyl    chloride in dichloromethane; and-   process e): reaction with N,N-diisopropylamine and 2-cyanoethyl    N,N-diisopropylphosphoroamidite in dichloromethane.

For details, refer to Examples described later.

The above process b) is a process for methylation of guanosine at theposition-8, in which a method described in Tetrahedron, 30, 2677-2682,1974 is employed, but the alkylation including the methylation ofguanosine at the position-8 can be performed by the following method.

That is, after a hydroxyl group in ribose is protected, for example,with a t-butyldimethylsilyl group, the position-8 of guanosine issubjected to lithiation with lithium diisopropylamine (LDA),subsequently reacted with a proper alkyl halide to selectively introducean alkyl group. Consequently, in case of introducing other alkyl group,this method can be applied (refer to Chem. Pharm. Bull. 35, 72-79,1987). Introduction of a methyl group can also be performed by thismethod.

A method for integrating guanosine having a lower alkyl group at theposition-8 into an oligonucleotide by using the guanosine derivative ofthe present invention, includes a solid-phase synthesis of DNA oligomerusing a commercially available DNA synthesizer. Each operationalprocedure can be sufficiently performed according to a protocol attachedto each of the DNA synthesizers.

The content of the specification of JP Application No. 2003-052815 isincorporated herein in its entirety.

EXAMPLES

The present invention will be explained more specifically by usingExamples, however, the present invention should not be construed to belimited by these Examples.

Example 1

Synthesis of8-methyl-N-isobutyryl-5′-O-dimethoxytrityl-2′-t-butyldimethylsilyl-guanosine-(2-cyanoethyl-N,N-diisopropylphosphoroamidite(Compound 5)(guanosine derivative of the general formula [1], wherein R¹is an isobutyryl group, R² is a Methyl Group, R³ is at-butyldimethylsilyloxy group and R⁴ is a cyanoethyl group)

(1) Synthesis of N-isobutyl-guanosine (Compound 1):

Guanosine (7 g, 25 mmol) was dried three times by evaporation ofpyridine and suspended in pyridine (140 ml). Trimethylsilyl chloride(17.5 ml, 125 mmol) was added thereto. After the solution was stirredfor 2 hours, isobutyric anhydride 21 ml (125 mmol) was added, and themixture was stirred for 4 hours at room temperature. After the reactionwas completed, the reaction mixture was added with water (35 ml) underice-cooling, and stirred for further 15 minutes. Then, the reactionmixture was added with 29% aqueous ammonia (35 ml) and stirred for 10minutes. After the solvent was removed off in vacuo, the residue wasextracted with dichloromethane 200 ml, and purified by the silica gelcolumn chromatography (developing solvent: methanol/dichloromethane=2/8)to obtain Compound 1 (3.6 g, yield: 39%).

NMR spectral data of thus obtained compound are shown below.

¹H NMR (500 MHz, DMSO-d₆) δ: 8.24 (s, 1H, H-8), 5.80 (m, 1H, 1′), 4.42(m, 1H, 2′), 3.88 (m, 4′), 4.08 (m, 1H, 3′), 3.33 (m, 2H, 5′), 2.77 (m,1H, isobutyryl CH), 1.12 (d, 6H, J=7 Hz, 2CH₃) ppm.

(2) Synthesis of 8-methyl-N-isobutyl-guanosine (Compound 2):

To a solution of H₂SO₄ (1N) 160 ml containing the Compound 1 (1 g, 2.6mmol) obtained in (1) and FeSO₄.7H₂O (6.7 g, 24.1 mmol), an aqueoussolution (100 ml) containing 70% t-butyl hydroperoxide (9.5 mmol) wasadded dropwise over a period of 5 minutes. After being stirred at 0° C.for 1 hour, the reaction mixture was neutralized with aqueous saturatedKOH solution. The supernatant obtained by centrifugation wasconcentrated in vacuo, and the residue was purified by the silica gelcolumn chromatography (developing solvent: methanol/dichloromethane=1/9)to obtain Compound 2 (0.4 g, yield: 41%).

NMR spectral data of thus obtained compound are shown below.

¹H NMR (500 MHz, DMSO-d₆) δ: 5.81 (m, 1H, 1′), 4.32 (m, 1H, 2′), 3.86(m, 4′), 4.12 (m, 1H, 3′), 3.29 (m, 2H, 5′), 2.78 (m, 1H, isobutyrylCH), 2.47 (s, 3H, −8 CH₃), 1.11 (d, 6H, J=7 Hz, 2CH₃) ppm.

(3) Synthesis of 8-methyl-N-isobutyryl-5′-O— dimethoxytrityl-guanosine(Compound 3):

The Compound 2 (500 mg, 2.4 mmol) obtained in (2) was dried three timesby evaporation of pyridine and suspended in pyridine (140 ml).Dimethoxytrityl chloride (DMTrCl, 700 mg, 3.7 mmol), triethylamine (0.3ml, 2.1 mmol) and 4-dimethylaminopyridine (4.2 mg, 0.035 mmol) wereadded thereto, and the reaction mixture was stirred at room temperatureovernight. After the reaction was completed, the reaction mixture wasadded with 5% aqueous solution of NaHCO₃ (50 ml) under ice-cooling, thereaction mixture was extracted with dichloromethane (60 ml), and theextract was purified by the silica gel column chromatography (developingsolvent: methanol/dichloromethane=3/97) to obtain Compound 3 (0.5 g,yield: 60%).

NMR spectral data of thus obtained compound are shown below.

¹H NMR (500 MHz, DMSO-d₆) δ: 5.81 (m, 1H, 1′), 4.32 (m, 1H, 2′), 3.86(m, 4′), 4.12 (m, 1H, 3′), 3.29 (m, 2H, 5′), 2.78 (m, 1H, isobutyrylCH), 2.47 (s, 3H, −8 CH₃), 1.11 (d, 6H, J=7 Hz, 2CH₃) ppm.

(4) Synthesis of8-methyl-N-isobutyryl-5′-O-dimethoxytrityl-2′-t-butyldimethylsilyl-guanosine(Compound 4):

To a dichloromethane solution (25 ml) containing the Compound 3 (1 g,1.4 mmol) obtained in (3) and imidazole (238 g, 3.5 mmol),2′-t-butyldimethylsilyl chloride (252 mg) was added, and the mixture wasstirred at 20° C. for 16 hours.

After the reaction was completed, 10% aqueous solution of NaHCO₃ (30 ml)was added to the reaction mixture. The reaction mixture was extractedwith dichloromethane (40 ml), and the extract was purified by the silicagel column chromatography (developing solvent: hexane/ethyl acetate=5/1)to obtain Compound 4 (0.3 g, yield: 30%).

NMR spectral data of thus obtained compound are shown below. ¹H NMR (500MHz, DMSO-d₆) δ: 7.20-7.39 (m, 9H, ph), 6.89 (d, 4H, J=8.5 Hz, ph), 4.96(m, 1H, 1′), 4.16 (m, 1H, 3′), 3.74 (s, 6H, 2CH₃), 3.18 (m, 2H, 5′),3.02 (m, 1H, 6′), 2.77 (m, 1H, isobutyryl CH), 2.14 (m, 1H, 2′), 2.05(m, 2H, 2″ and 4′), 1.88 (s, 3H, −8 CH₃), 1.62 (m, 1H, 6″), 1.10 (d, 6H,J=7 Hz, 2CH₃) ppm.

(5) Synthesis of8-methyl-N-isobutyryl-5′-O-dimethoxytrityl-2′-t-butyldimethylsilyl-guanosine-(2-cyanoethyl-N,N-diisopropylphosphroamidite(Compound 5):

The Compound 4 (300 ml, 0.4 mmol) obtained in (4) was sealed in arubber-sealed bottle. After sealing the bottle, dry acetonitrile (3 ml)was added thereto, and inside of the bottle was evacuated by reducedpressure through a needle to evaporate acetonitrile and remove water byazeotrope. Dry dichloromethane (3 ml) was injected by a syringe, and dryN,N-diisopropylamine (0.45 ml, 0.6 mmol) and2-cyanoethyl-N,N-diisopropylphosphoroamidite (0.25 ml, 1 mmol) wereadded thereto. The reaction mixture was stirred at room temperatureovernight. After 10% aqueous NaHCO₃ solution (10 ml) was added, thereaction mixture was extracted with dichloromethane (20 ml), and theorganic layer was dried over Na₂SO₄. The extract was purified by thesilica gel column chromatography (developing solvent:hexane/acetone/triethylamine=49/49/2) to obtain Compound 5 (240 mgyield: 60%).

Spectral data of thus obtained compound are shown below.

ESIMS (m/e) as C₅₂H₇₅N₇₀₉PSi: Calculated: (M+H) 1000.5. Observed:1000.8. (Measured by using PE SCIEX API 165)

Example 2 Solid Phase Syntheses of DNA Oligomers

The compound 5 (200 ml, 0.2 mmol) obtained in Example 1 was sealed in arubber-sealed bottle. Dry acetonitrile (2 ml) was added thereto andevaporated in vacuo. The residue was dissolved by adding dryacetonitrile (0.3 ml), and the solution was supplied to the OLIGO 1000DNA synthesizer (Beckman Inc.), then oligonucleotide d (CGCMerGCG), d(CMerGCACMerGCG) and d (CMerGCATMerGTG) were synthesized according tothe protocol attached to the DNA synthesizer.

Compositions and concentrations of the synthesized oligonucleotides wereconfirmed by enzymatic hydrolysis to mononucleosides.

In the sequences of the above oligonucleotides, MerG represents8-methylguanosine.

Example 3 Circular Dichroism Experiments

Circular dichroism spectra of three types of oligonucleotides obtainedin Example 2 were analyzed using the circular dichroismspectrophotometer (AVIV MODEL 62 DS/202 CD spectrophotometer, AVIVBiomedical Inc.). CD (circular dichroism) spectra of theoligonucleotides (concentration: 0.15 mM base conc., 5 mM sodiumcacodylate buffer, pH 7.0) were measured by using a cell, 1 cm inlength, in various concentrations of NaCl. Results are shown in FIG. 1.

FIG. 1 (a) shows CD spectra of oligonucleotide 5′-CGCMerGCG-3′; FIG. 1(b) shows CD spectra of oligonucleotide 5′-CMerGCACMerGCG-3′; and FIG. 1(c) shows CD spectra of oligonucleotide 5′-CMerGCATMerGTG-3′.

Further, a NaCl concentration at the midpoint of B-Z transition of eacholigonucleotide was obtained by measuring CD spectra with theconcentration of NaCl varied. Results are shown in Table 1. Forcomparison, an oligonucleotide, to which 8-methyldeoxyguanosine (MeG) isintroduced in place of 8-methylguanosine (MerG), was measured similarly,and the results are also shown in Table 1.

In Table 1, MeG represents 8-methyldeoxyguanosine and MerG represents8-methylguanosine.

TABLE 1 Experiment 1 d(CGCGCG) 2.6 M Experiment 2 d(CGCMeGCG) 30 mMExperiment 3 d(CGCMerGCG) <5 mM Experiment 4 d(CGCGTGCG)/d(CMeGCACMeGCG)800 mM Experiment 5 d(CGCGTGCG)/d(CMerGCACMerGCG) 50 mM Experiment 6d(CMeGCATMeGTG)/d(GCGTACAC) 2450 mM Experiment 7d(CMerGCATMerGTG)/d(GCGTACAC) 200 mM

As apparent from Table 1, it can be understood that the integration of8-methylguanosine into various oligonucleotides results in significantlowering of the midpoint in any case, showing stabilization of Z-formDNA even in very low concentration of salt. The reason may be that whena methyl group is introduced in guanosine at the position-8, the basethereof has a tendency to adopt a syn orientation, and the introductionof a hydroxyl group at the position-2′ results in an increase ofhydrophilicity, and puckering of sugar is further stabilized by adoptinga C3′-endo conformation.

INDUSTRIAL APPLICABILITY

The present invention provides 8-methylguanosine, which is a novelmonomer unit capable of stabilizing Z-form DNA more effectively, aguanosine derivative represented by the general formula [1], which is areagent for integrating the monomer unit into an oligonucleotide, and amethod for stabilizing Z-form DNA by using the reagent. Since8-methylguanosine of the present invention, which is a compound preparedby introducing a methyl group in guanosine at the position-8, canstabilize Z-form DNA more potently by several times as compared with theconventionally known 8-methyldeoxyguanosine (MeG), it can be predictableto be an excellent tool for studying the Z-form DNA.

1. A method for stabilizing Z-form DNA, comprising synthesizing DNA witha guanosine derivative represented by general formula [1]:

wherein, R¹ represents an acyl group, R² represents a lower alkyl group,R³ represents a protected hydroxyl group, R⁴ represents a cyanoethylgroup or an allyl group, and DMTr represents a dimethoxytrityl group. 2.The method of claim 1, wherein the DNA is synthesized using solid-phasesynthesis.